Photoimmunotechnology as a powerful biological tool for molecular-based elimination of target cells and microbes, including bacteria, fungi and viruses.

Microbial pathogens, including bacteria, fungi and viruses, can develop resistance to clinically used drugs; therefore, finding new therapeutic agents is an ongoing challenge. Recently, we reported the photoimmuno-antimicrobial strategy (PIAS), a type of photoimmunotechnology, that enables molecularly targeted elimination of a wide range of microbes, including the viral pathogen severe acute respiratory syndrome coronavirus 2 and the multidrug-resistant bacterial pathogen methicillin-resistant Staphylococcus aureus (MRSA). PIAS works in the same way as photoimmunotherapy (PIT), which has been used to treat recurrent head and neck cancer in Japan since 2020. Both PIAS and PIT use a monoclonal antibody conjugated to a phthalocyanine derivative dye that undergoes a shape change when photoactivated. This shape change induces a structural change in the antibody-dye conjugate, resulting in physical stress within the binding sites of the conjugate and disrupting them. Therefore, targeting accuracy and flexibility can be determined based on the specificity of the antibody used. In this protocol, we describe how to design a treatment strategy, label monoclonal antibodies with the dye and characterize the products. We provide detailed examples of how to set up and perform PIAS and PIT applications in vitro and in vivo. These examples are PIAS against microbes using MRSA as a representative subject, PIAS against viruses using severe acute respiratory syndrome coronavirus 2 in VeroE6/TMPRSS2 cells, PIAS against MRSA-infected animals, and in vitro and in vivo PIT against cancer cells. The in vitro and in vivo protocols can be completed in ~3 h and 2 weeks, respectively.

Antimicrobial strategy for targeted elimination of different microbes, including bacterial, fungal and viral pathogens.

The continuous emergence of microbial pathogens for which there are no effective antimicrobials threatens global health, necessitating novel antimicrobial approaches. Here, we present a targeted antimicrobial strategy that can be applied to various microbial pathogens. A photoimmuno-conjugate composed of an antibody against the target pathogen and a photoplastic phthalocyanine-derivative probe that generates photo-induced mechanical stress was developed based on photoimmuno-technology. This strategy, named as photoimmuno-antimicrobial strategy (PIAS), eliminates targeted pathogens, regardless of the target species or drug-resistance status. Specifically, PIAS acts on a broad range of microbes, including the bacterial pathogen Staphylococcus aureus, fungal pathogen Candida albicans, including their drug-resistant strains, and viral pathogen SARS-CoV-2, the causative agent of COVID-19. Furthermore, PIAS protects mice from fatal infections without damaging the non-targeted host microbiota and tissues. This study may contribute to the development of next-generation anti-infective therapies.

TAS-119, a novel selective Aurora A and TRK inhibitor, exhibits antitumor efficacy in preclinical models with deregulated activation of the Myc, ß-Catenin, and TRK pathways.

Aurora kinase A, a mitotic kinase that is overexpressed in various cancers, is a promising cancer drug target. Here, we performed preclinical characterization of TAS-119, a novel, orally active, and highly selective inhibitor of Aurora A. TAS-119 showed strong inhibitory effect against Aurora A, with an IC50 value of 1.04 nmol/L. The compound was highly selective for Aurora A compared with 301 other protein kinases, including Aurora kinase B. TAS-119 induced the inhibition of Aurora A and accumulation of mitotic cells in vitro and in vivo. It suppressed the growth of various cancer cell lines harboring MYC family amplification and CTNNB1 mutation in vitro. In a xenograft model of human lung cancer cells harboring MYC amplification and CTNNB1 mutation, TAS-119 showed a strong antitumor activity at well-tolerated doses. TAS-119 induced N-Myc degradation and inhibited downstream transcriptional targets in MYCN-amplified neuroblastoma cell lines. It also demonstrated inhibitory effect against tropomyosin receptor kinase (TRK)A, TRKB, and TRKC, with an IC50 value of 1.46, 1.53, and 1.47 nmol/L, respectively. TAS-119 inhibited TRK-fusion protein activity and exhibited robust growth inhibition of tumor cells via a deregulated TRK pathway in vitro and in vivo. Our study indicates the potential of TAS-119 as an anticancer drug, especially for patients harboring MYC amplification, CTNNB1 mutation, and NTRK fusion.

Futibatinib Is a Novel Irreversible FGFR 1-4 Inhibitor That Shows Selective Antitumor Activity against FGFR-Deregulated Tumors.

FGFR signaling is deregulated in many human cancers, and FGFR is considered a valid target in FGFR-deregulated tumors. Here, we examine the preclinical profile of futibatinib (TAS-120; 1-[(3S)-[4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3, 4-d] pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one), a structurally novel, irreversible FGFR1-4 inhibitor. Among a panel of 296 human kinases, futibatinib selectively inhibited FGFR1-4 with IC50 values of 1.4 to 3.7 nmol/L. Futibatinib covalently bound the FGFR kinase domain, inhibiting FGFR phosphorylation and, in turn, downstream signaling in FGFR-deregulated tumor cell lines. Futibatinib exhibited potent, selective growth inhibition of several tumor cell lines (gastric, lung, multiple myeloma, bladder, endometrial, and breast) harboring various FGFR genomic aberrations. Oral administration of futibatinib led to significant dose-dependent tumor reduction in various FGFR-driven human tumor xenograft models, and tumor reduction was associated with sustained FGFR inhibition, which was proportional to the administered dose. The frequency of appearance of drug-resistant clones was lower with futibatinib than a reversible ATP-competitive FGFR inhibitor, and futibatinib inhibited several drug-resistant FGFR2 mutants, including the FGFR2 V565I/L gatekeeper mutants, with greater potency than any reversible FGFR inhibitors tested (IC50, 1.3-50.6 nmol/L). These results indicate that futibatinib is a novel orally available, potent, selective, and irreversible inhibitor of FGFR1-4 with a broad spectrum of antitumor activity in cell lines and xenograft models. These findings provide a strong rationale for testing futibatinib in patients with tumors oncogenically driven by FGFR genomic aberrations, with phase I to III trials ongoing. SIGNIFICANCE: Preclinical characterization of futibatinib, an irreversible FGFR1-4 inhibitor, demonstrates selective and potent antitumor activity against FGFR-deregulated cancer cell lines and xenograft models, supporting clinical evaluation in patients with FGFR-driven tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4986/F1.large.jpg.

Acquired resistance to trastuzumab/pertuzumab or to T-DM1 in vivo can be overcome by HER2 kinase inhibition with TAS0728.

HER2-targeting antibodies (trastuzumab, pertuzumab) and a HER2-directed antibody-drug conjugate (trastuzumab emtansine: T-DM1) are used for the treatment of HER2-overexpressing breast cancer. However, these treatments eventually become ineffective due to acquired resistance and there is an urgent need for alternative therapies. TAS0728 is a small-molecule, irreversible selective HER2 kinase inhibitor. In the present study, we established new in vivo models of cancer resistance by continuous exposure to a combination of trastuzumab and pertuzumab or to T-DM1 for evaluating the effect of TAS0728 on HER2 antibody-resistant populations. Treatment with trastuzumab and pertuzumab or with T-DM1 initially induced tumor regression in NCI-N87 xenografts. However, tumor regrowth during treatment indicated loss of drug effectiveness. In tumors with acquired resistance to trastuzumab and pertuzumab or to T-DM1, HER2-HER3 phosphorylation was retained. Switching to TAS0728 resulted in a significant anti-tumor effect associated with HER2-HER3 signal inhibition. No alternative receptor tyrosine kinase activation was observed in these resistant tumors. Furthermore, in a patient-derived xenograft model derived from breast cancer refractory to both trastuzumab/pertuzumab and T-DM1, TAS0728 exerted a potent anti-tumor effect. These results suggest that tumors with acquired resistance to trastuzumab and pertuzumab and to T-DM1 are still dependent on oncogenic HER2-HER3 signaling and are vulnerable to HER2 signal inhibition by TAS0728. These results provide a rationale for TAS0728 therapy for breast cancers that are refractory to established anti-HER2 therapies.

Cancer neovasculature-targeted near-infrared photoimmunotherapy (NIR-PIT) for gastric cancer: different mechanisms of phototoxicity compared to cell membrane-targeted NIR-PIT.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) constitutes a new class of molecular-targeted theranostics utilizing monoclonal antibody (mAb)-photosensitizer conjugates and NIR light. In this study, we developed a new type of NIR-PIT targeting vascular endothelial growth factor receptor 2 (VEGFR-2) expressed on vascular endothelium in an experimental gastric cancer model and evaluated the feasibility by comparing conventional NIR-PIT targeting cancer cell membrane in vitro and in vivo. METHODS: HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. Anti-HER2 mAb, trastuzumab and anti-VEGFR-2 mAb, DC101 were conjugated to photosensitizer, IR700. Phototoxicity in response to NIR-PIT were investigated in vitro and in vivo. Microvessel densities, as an indicator of angiogenesis, were counted in harvested xenografts after NIR-PIT to elucidate the mechanism. RESULTS: DC101-IR700 did not induce phototoxic effect in vitro because of the absence of expression of VEGFR-2 in NCI-N87 cancer cells. However, it induced an antitumor effect in NCI-N87 xenograft tumors accompanied with damage in tumor neovasculature as determined by decreasing tumor microvessel density, which represents a different mechanism than that of conventional NIR-PIT targeting antigens expressed on the tumor cell membrane. CONCLUSION: We demonstrated a new approach of NIR-PIT utilizing a target on vascular endothelium, such as VEGFR-2, and this treatment might lead to the development of a new therapeutic strategy for human gastric cancer.

TAS-121, A Selective Mutant EGFR Inhibitor, Shows Activity Against Tumors Expressing Various EGFR Mutations Including T790M and Uncommon Mutations G719X.

TAS-121 is a novel orally active selective covalent inhibitor of the mutant EGFR. We performed preclinical characterization of TAS-121 and compared its efficacy and selectivity for common EGFR mutations (Ex19del and L858R), first- and second- generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) resistance mutation (T790M), and uncommon mutations (G719X and L861Q) with those of other EGFR-TKIs. We also commenced investigation of the clinical benefits of TAS-121. The IC50 for intracellular EGFR phosphorylation was determined by using Jump-In GripTite HEK293 cells transiently transfected with EGFR expression vectors. Mouse xenograft models were used to evaluate the antitumor activity of TAS-121. TAS-121 potently inhibited common activating and resistance EGFR mutations to the same extent as another third-generation EGFR-TKI (osimertinib). In addition, TAS-121 showed equivalent inhibitory activity against some uncommon mutations such as G719X and L861Q. Furthermore, TAS-121 demonstrated greater selectivity for mutant EGFRs versus the wild-type EGFR compared with other EGFR-TKIs. Moreover, TAS-121 displayed antitumor activity in SW48 (EGFR G719S) and NCI-H1975 (EGFR L858R/T790M) xenograft models, and achieved an objective response in patients with NSCLC with EGFR mutations including G719A mutation. In conclusion, TAS-121 is a novel third-generation EGFR-TKI and demonstrates antitumor activities in patients with NSCLC expressing either common or uncommon EGFR mutations.

TAS0728, A Covalent-binding, HER2-selective Kinase Inhibitor Shows Potent Antitumor Activity in Preclinical Models.

Activated HER2 is a promising therapeutic target for various cancers. Although several reports have described HER2 inhibitors in development, no covalent-binding inhibitor selective for HER2 has been reported. Here, we report a novel compound TAS0728 that covalently binds to HER2 at C805 and selectively inhibits its kinase activity. Once TAS0728 bound to HER2 kinase, the inhibitory activity was not affected by a high ATP concentration. A kinome-wide biochemical panel and cellular assays established that TAS0728 possesses high specificity for HER2 over wild-type EGFR. Cellular pharmacodynamics assays using MCF10A cells engineered to express various mutated HER2 genes revealed that TAS0728 potently inhibited the phosphorylation of mutated HER2 and wild-type HER2. Furthermore, TAS0728 exhibited robust and sustained inhibition of the phosphorylation of HER2, HER3, and downstream effectors, thereby inducing apoptosis of HER2-amplified breast cancer cells and in tumor tissues of a xenograft model. TAS0728 induced tumor regression in mouse xenograft models bearing HER2 signal-dependent tumors and exhibited a survival benefit without any evident toxicity in a peritoneal dissemination mouse model bearing HER2-driven cancer cells. Taken together, our results demonstrated that TAS0728 may offer a promising therapeutic option with improved efficacy as compared with current HER2 inhibitors for HER2-activated cancers. Assessment of TAS0728 in ongoing clinical trials is awaited (NCT03410927).

Near-Infrared Photochemoimmunotherapy by Photoactivatable Bifunctional Antibody-Drug Conjugates Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy based on antibody-photoabsorber conjugates and NIR light irradiation. Recent studies have shown effective tumor control, including that of human epidermal growth factor receptor 2 (HER2)-positive cancer, by selective molecular targeting with NIR-PIT. However, the depth of NIR light penetration limits its use. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent maytansinoid DM1. Here, we developed bifunctional antibody-drug-photoabsorber conjugates, T-DM1-IR700, that can work as both NIR-PIT and chemoimmunotherapy agents. We evaluated the feasibility of T-DM1-IR700-mediated NIR light irradiation by comparing the in vitro and in vivo cytotoxic efficacy of trastuzumab-IR700 (T-IR700)-mediated NIR light irradiation in HER2-expressing cells. T-IR700 and T-DM1-IR700 showed almost identical binding to HER2 in vitro and in vivo. Owing to the presence of internalized DM1 in the target cells, NIR-PIT using T-DM1-IR700 tended to induce greater cytotoxicity than that of NIR-PIT using T-IR700 in vitro. In vivo NIR-PIT using T-DM1-IR700 did not show a superior antitumor effect to NIR-PIT using T-IR700 in subcutaneous small-tumor models, which could receive sufficient NIR light. In contrast, NIR-PIT using T-DM1-IR700 tended to reduce the tumor volume and showed significant prolonged survival compared to NIR-PIT using T-IR700 in large-tumor models that could not receive sufficient NIR light. We successfully developed a T-DM1-IR700 conjugate that has a similar immunoreactivity to the parental antibody with increased cytotoxicity due to DM1 and potential as a new NIR-PIT agent for targeting tumors that are large and inaccessible to sufficient NIR light irradiation to activate the photoabsorber IR700.

Combination photoimmunotherapy with monoclonal antibodies recognizing different epitopes of human epidermal growth factor receptor 2: an assessment of phototherapeutic effect based on fluorescence molecular imaging.

Photoimmunotherapy is a new class of molecular targeted cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer and irradiation with near-infrared (NIR) light for both imaging and therapy. Here, we sought to determine the feasibility of combining photoimmunotherapy using conjugates of human epidermal growth factor receptor 2 (HER2)-specific mAb-photosensitizer IR700, trastuzumab-IR700 and pertuzumab-IR700. HER2-expressing and non-expressing cells were treated with mAb-IR700 conjugates and irradiated with NIR light. Fluorescence imaging and cytotoxic effects were examined in cultured HER2-expressng cancer cell lines and in a mouse tumor xenograft model. Trastuzumab-IR700 and pertuzumab-IR700 could specifically bind to HER2 without competing, and the combination treatment of both agents yielded stronger HER2-specific IR700 fluorescence signals than with the treatment with either agent singly. A cytotoxicity assay showed that the combination treatment of both trastuzumab-IR700 and pertuzumab-IR700 followed by NIR light irradiation induced stronger cytotoxic effect than with treatment of either agent plus NIR light irradiation. Furthermore, the phototoxic and cytotoxic effects of mAb depended on HER2-specific IR700 signal intensities. Consistent with in vitro studies, in xenograft tumor models also, IR700 fluorescence imaging-guided NIR light irradiation after the combination treatment of trastuzumab-IR700 and pertuzumab-IR700 led to stronger antitumor effects than by treatment with either agent followed by NIR light irradiation. In conclusion, fluorescence molecular imaging can facilitate the assessment of treatment outcomes of molecular targeted photoimmunotherapy, which holds great potential in facilitating better outcomes in cancer patients.

Molecular targeted photoimmunotherapy for HER2-positive human gastric cancer in combination with chemotherapy results in improved treatment outcomes through different cytotoxic mechanisms.

BACKGROUND: Photoimmunotherapy (PIT) is a novel type of molecular optical imaging-guided cancer phototherapy based on a monoclonal antibody conjugated to a photosensitizer, IR700, in combination with near-infrared (NIR) light. PIT rapidly causes target-specific cell death by inducing cell membrane damages and appears to be highly effective; however, we have previously demonstrated that tumor recurrences were eventually seen in PIT-treated mice, likely owing to inhomogeneous mAb-IR700 conjugate distribution in the tumor, thus limiting the effectiveness of PIT as a monotherapy. Here, we examined the effects of human epidermal growth factor-2 (HER2)-targeted PIT in combination with 5-fluorouracil (5-FU) compared to PIT alone for HER2-expressing human gastric cancer cells. METHODS: NCI-N87 cells, HER2-positive human gastric cancer cells, were used for the experiments. Trastuzumab, a monoclonal antibody directed against HER2, was conjugated to IR700. To assess the short-term cytotoxicity and examine the apoptotic effects upon addition of 5-FU in vitro, we performed LIVE/DEAD and caspase-3 activity assays. Additionally, to explore the effects on long-term growth inhibition, trypan blue dye exclusion assay was performed. NCI-N87 tumor xenograft models were prepared for in vivo treatment studies and the tumor-bearing mice were randomized into various treatment groups. RESULTS: Compared to PIT alone, the combination of HER2-targeted PIT and 5-FU rapidly induced significant cytotoxicity in both the short-term and long-term cytotoxicity assays. While both 5-FU and/or trastuzumab-IR700 conjugate treatment induced an increase in caspase-3 activity, there was no additional increase in caspase-3 activity upon NIR light irradiation after incubation with 5-FU and/or trastuzumab-IR700. The combination of HER2-targeted PIT and 5-FU resulted in greater and longer tumor growth inhibition than PIT monotherapy in vivo. This combined effect of PIT and 5-FU is likely owing to their different mechanisms of inducing tumor cell death, namely necrotic membrane damage by PIT and apoptotic cell death by 5-FU and trastuzumab. CONCLUSIONS: PIT in combination with 5-FU resulted in enhanced antitumor effects compared to PIT alone for HER2-expressing human gastric cancer in vitro and in vivo. This combination photoimmunochemotherapy represents a practical method for treating human gastric cancer and should be investigated further in the clinical setting.

The novel VEGF receptor/MET-targeted kinase inhibitor TAS-115 has marked in vivo antitumor properties and a favorable tolerability profile.

VEGF receptor (VEGFR) signaling plays a key role in tumor angiogenesis. Although some VEGFR signal-targeted drugs have been approved for clinical use, their utility is limited by associated toxicities or resistance to such therapy. To overcome these limitations, we developed TAS-115, a novel VEGFR and hepatocyte growth factor receptor (MET)-targeted kinase inhibitor with an improved safety profile. TAS-115 inhibited the kinase activity of both VEGFR2 and MET and their signal-dependent cell growth as strongly as other known VEGFR or MET inhibitors. On the other hand, kinase selectivity of TAS-115 was more specific than that of sunitinib and TAS-115 produced relatively weak inhibition of growth (GI50 > 10 µmol/L) in VEGFR signal- or MET signal-independent cells. Furthermore, TAS-115 induced less damage in various normal cells than did other VEGFR inhibitors. These data suggest that TAS-115 is extremely selective and specific, at least in vitro. In in vivo studies, TAS-115 completely suppressed the progression of MET-inactivated tumor by blocking angiogenesis without toxicity when given every day for 6 weeks, even at a serum-saturating dose of TAS-115. The marked selectivity of TAS-115 for kinases and targeted cells was associated with improved tolerability and contributed to the ability to sustain treatment without dose reduction or a washout period. Furthermore, TAS-115 induced marked tumor shrinkage and prolonged survival in MET-amplified human cancer-bearing mice. These data suggest that TAS-115 is a unique VEGFR/MET-targeted inhibitor with improved antitumor efficacy and decreased toxicity.

Carnitine/organic cation transporter OCTN2 (Slc22a5) is responsible for renal secretion of cephaloridine in mice.

Carnitine/organic cation transporter (OCTN) 2 (SLC22A5) plays a pivotal role in renal tubular reabsorption of carnitine, a vitamin-like compound, on apical membranes of proximal tubules, but its role in relation to therapeutic drugs remains to be clarified. The purpose of the present study was to elucidate the involvement of OCTN2 in renal disposition of a beta-lactam antibiotic, cephaloridine (CER), based on experiments with juvenile visceral steatosis (jvs) mice, which have a functional deficiency of the octn2 gene. Renal clearance of CER during constant intravenous infusion in wild-type mice was much higher than could be accounted for by glomerular filtration, but was decreased by increasing the infusion rate with minimal change in kidney-to-plasma concentration ratio, suggesting the existence of saturable transport mechanism(s) across the apical membranes. The plasma concentration profile and kidney-to-plasma concentration ratio after intravenous injection in jvs mice were higher than those in wild-type mice, whereas renal clearance in jvs mice was much lower than that in wild-type mice and could be accounted for by glomerular filtration. Uptake of CER by mouse OCTN2 was shown in Xenopus laevis oocytes expressing mouse OCTN2. The CER transport by OCTN2 exhibited saturation with K(m) of approximately 3 mM, which is similar to the renal CER concentration exhibiting saturation in renal clearance in vivo. The OCTN2-mediated CER transport was inhibited by carnitine and independent of Na(+) replacement in the medium. These results show OCTN2 on apical membranes of proximal tubules plays a major role in renal secretion of CER in mice.

L-type fatty acid binding protein transgenic mouse as a novel tool to explore cytotoxicity to renal proximal tubules.

A novel biomarker of renal dysfunction, liver-type fatty acid binding protein (L-FABP), which is expressed in human proximal tubules, binds to lipid peroxidation products during renal injury and is excreted into the urine. Here, we examined the usefulness of human L-FABP transgenic (Tg) mice as a tool to explore nephrotoxicity, employing two model drugs, cephaloridine and cisplatin, which are taken up by renal tubules via organic anion and cation transporters, respectively. Urinary excretion of L-FABP increased after administration of cephaloridine in most of the Tg mice, whereas glomerular filtration markers such as blood-urea-nitrogen (BUN) and plasma creatinine (CRE) were almost unchanged. Thus, L-FABP is a highly sensitive detector of the nephrotoxicity of cephaloridine. Urinary excretion of L-FABP in the Tg mice also increased after administration of cisplatin, and this increase was reduced by coadministration of cimetidine. Both BUN and CRE also increased after the cisplatin treatment, but these parameters were minimally affected by coadministration of cimetidine, suggesting that cimetidine reduces cisplatin-induced renal tubular toxicity with only a minimal effect on the glomerulus. These results indicate that the L-FABP Tg mouse should be a useful drug screening system to evaluate specifically the toxicity of transporter substrates to renal tubules.